Proteins that play a role in the herpesviral DNA encapsidation process (the cleaving and packaging of viral DNA into capsids) have become promising novel antiviral targets. A series of non-nucleoside compounds that inhibit VZV DNA encapsidation have been described. N-?-methylbenzyl-N'-aryl thiourea analogs were demonstrated to have specific and potent activity against VZV. The ?-methylbenzyl compounds are novel in that they are the first agents to target the DNA cleavage and packaging mechanism of VZV and are currently the only inhibitors reported to target a mechanism different from inhibition of the VZV DNA polymerase. An understanding of such mechanisms for human pathogens has the added benefit of the identification of novel targets for antiviral chemotherapy. The proposed studies on the proteins involved in the VZV DNA encapsidation process are a continuation of the research program initiated in the previously funded application. The experiments proposed in the specific aims have the potential to better clarify our understanding of the interactions and functional properties of proteins involved in the process of DNA cleavage and packaging for VZV, and for the Herpesviridae in general. This is significant with respect to the development of novel antiviral therapies since previous studies have shown that it is feasible to identify chemical inhibitors that specifically target the encapsidation process of herpesviruses. During the last funding period, our laboratory cloned and sequenced all seven of the putative VZV DNA encapsidation genes. Antisera were produced to six of the seven putative DNA encapsidation gene products. Using the antisera, polypeptides corresponding to VZV ORFs 25, 30, 34, 43, 45/42 and 54 were identified in VZV infected cells. Numerous protein-protein interactions were identified via GST pull-down, yeast two-hybrid, and co-immunoprecipitation. This work was done primarily by student researchers at IPFW. More than 25 students received training in my laboratory during the previous funding period. A continuation of the original project includes experiments proposed to more specifically characterize the terminase complex and include (i) mapping interacting protein-protein domains between pORF25, pORF30, and pORF45/42, (ii) production of stably transfected cell lines to be used for the isolation of the first set of VZV encapsidation mutant viruses, and (iii) isolation and characterization of the VZV pORF54 portal protein using a recently isolated pORF54 expressing baculovirus. Additionally, preliminary evidence suggests that mutations conferring resistance to a previously described ?-methylbenzyl inhibitor series cluster in an important functional region of pORF54. We propose to isolate an extensive panel of drug resistant isolates to further define the interactions between pORF54 and the inhibitors. The results may yield clues to both portal structure and a binding site for the ?- methylbenzyl inhibitor series. In support of the spirit of NIH AREA grants, these studies are once again designed to provide extensive laboratory experience to student researchers. PUBLIC HEALTH RELEVANCE: There are eight different human herpesviruses (Herpes simplex types 1 and 2, Varicella-zoster virus, Epstein-Barr virus, Human Cytomegalovirus, Human herpesvirus types 6 and 7, and Kaposi's Sarcoma virus) that can cause mild to severe disease. Efforts over the last decade have focused on the identification and development of improved therapies including less toxic compounds with novel mechanisms of action. Our laboratory focuses on understanding new strategies to inhibit herpesvirus replication, namely, on a series of novel drug candidates that prevent infectious viral particles from forming in infected cells.